Bacteria belonging to the genus Bacillus have attractive properties for the production of industrially important enzymes. They are safe, noninfectious to humans, do not produce toxic substances, and can easily secrete gene products into the culture broth, unlike E. coli and yeasts.
Alpha-amylase is one of the industrially most important enzymes and is used in large amounts, for example, in the textile and food industries, for the hydrolysis of starch. The industrial production of alpha-amylase is thus of substantial economic importance.
In the manufacture of alpha-amylase, it is generally known that by fermentation with different types of microorganisms of the genus Bacillus, including Bacillus subtilis and its variants, alpha-amylase can be obtained in commercially useful amounts. It is particularly advantageous if the fermentation medium itself already contains high concentrations of the active enzyme. There has, therefore, been much to apply new cultivation and processing techniques to obtain the highest possible enzyme concentration in the fermentation medium. For example, Yoneda et al. in Applied And Environmental Microbiology (Vol 39) 274 et seq. (1980); Molecular Cloning And Gene Regulation In Bacilli (Ganesan et al. eds 1982) have described a process using a strain of B. subtilis which is employed in industry and has been modified by means of a specific transfer of gene fragments to increase the yield of alpha amylase in the fermentation liquor from 130-150 alpha-amylase units/ml to 25,000 alpha-amylase units/ml, the term “alpha-amylase units” having an art-recognized meaning set out in detail below. This increase represents an improvement by a factor of about 170 in the production of alpha-amylase. Such an increase in productivity is, however, still insufficient for the industrial production of alpha-amylase.
Currently, by virtue of the progress of gene recombination technology and plasmid expression vectors in B. subtilis, alpha-amylase genes have been inserted into plasmid expression vectors for over-expression. As host bacteria for producing alpha-amylases of the foreign alpha amylase genes in such gene recombination technology, strains of B. subtilis are widely used because their biological characteristics have been sufficiently investigated and further, they have no known pathogenicity and can easily grow in culture media having relatively simple compositions. However, the use of plasmid systems require the addition of antibiotics to the culture medium for maintaining the stability of the plasmid, see, for example, Wei et al., Biotechnology and Bioengineering 33, 1010-1020 (1989). Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous applications of antibiotic selection pressure owing to segregational plasmid instability. The use of antibiotics is expensive for industrial scale production. The presence of antibiotics also increases the cost of the down stream processing as the presence of antibiotics in the final product is not acceptable in most food applications.
An important application for phage expression vectors lies in the overexpression of cloned genes for purposes of protein purification. The prophage-based vectors have the advantages of increased stability because of their chromosomal locations and convenient regulation provided by the phage immunity system. Moreover, phage induction, which can be controlled by temperature shift in appropriate mutants, results not only in expression from strong phage promoters but also in an increased copy number through phage DNA replication.
Expression vectors have been developed from the temperature phage φ105, which is present in most of the genetic strains of B. subtilis, see Seaman, E et al. 1964, Biochemistry 3, 607-613. Several modifications have been incorporated in the prophage: (i) a mutation rendering the prophage temperature inducible, (ii) the identification of a suitable cloning site within a strongly induced transcription unit, and (iii) a mutation preventing lysis of the host cell. Clearly, for a secreted gene product, it is desirable that the host cell does not lyse so that product can be purified from the cell supernatant. Vectors for high level protein production have been developed recently, mainly by the identification and use of a strong φ105 promoter. φ105MU209 was isolated as a colony exhibiting extremely strong expression of beta-galactosidase amount transformants generated by random insertion of a lacZ reporter gene into a temperature-inducible φ105 prophage, see Errington, J 1986. P. 217-227, in A T Ganesan and J A Hoch (ed.), Bacillus Molecular Genetics and Biotechnology Applications, Academic Press, Inc, Orlando, Fla. The structure of the insertion in φ105MU209 is complex. However, it is clear that the insertion has not only conferred strong inducible expression of lacZ following temperature induction but has also blocked host cell lysis that normally follows phage induction. The lacZ gene lies downstream from the strong dual phage promoters, see Leung, Y C and Errington, J, 1995. Gene 154, 1-6, that can be induced about 100-fold by thermoinduction of the prophage, which carries the cts-52 mutation. To facilitate the expression of other proteins in this system, the lacZ gene and part of the chloramphenicol resistance gene have been replaced with an erythromycin resistance gene. The resulting prophage expression vector was named as φ105MU331, Thornewell, S J et at, 1993, Gene 133, 47-53.
The method of homologous recombination has been used to replace the erythromycin resistance gene with heterologous genes, which are then overexpressed following thermoinduction. This system has been used to produce the secreted Bacillus cereus beta-lactamase I, with yields of about 0.5 g/l. The constructions are stable in the absence of selection (before induction), and it seems likely that phage DNA replication and the resultant increase in copy number contribute to the high level of expression.